Optimization of an anti-HIV hairpin ribozyme by in vitro selection

We have applied in vitro selection methods to achieve a large increase in the catalytic activity of a hairpin ribozyme targeted against a highly conserved 14-nucleotide sequence within HIV-1 pol RNA. The substrate specificity was changed by mutating 8 bases within the substrate-binding domain of the parental (-)sTRSV ribozyme. The resulting enzyme cleaved the HIV substrate specifically but with a 20-fold reduction in catalytic efficiency (kcat/KM). Following random mutagenesis, ribozymes with increased activity against the target sequence were selected through 10 rounds of in vitro selection. Selective pressure was increased by decreasing MgCl2 and spermidine concentrations, and reducing reaction time. Variant ribozymes with base substitutions A11-->G and U39-->C were selected in the population. These mutations were introduced singly and in combination into the trans-acting anti-HIV ribozyme. Each of the single-base substitutions significantly increased ribozyme activity, while the activity of double mutant was increased to nearly the level of the parental ribozyme. These findings demonstrate that in vitro selection is a powerful and efficient method to optimize ribozymes for the catalytic inactivation of targeted RNA molecules.